2493 Environmental Toxicology and Chemistry, Vol. 19, No. 10, pp. 2493–2501, 2000  2000 SETAC Printed in the USA 0730-7268/00 $9.00 + .00 FIXED-EFFECT-LEVEL TOXICITY EQUIVALENTS—A SUITABLE PARAMETER FOR ASSESSING ETHOXYRESORUFIN-O-DEETHYLASE INDUCTION POTENCY IN COMPLEX ENVIRONMENTAL SAMPLES WERNER BRACK,*² H ELMUT SEGNER,² M ONIKA MO ¨ DER,‡ and GERRIT SCHU ¨ U ¨ RMANN² ²Department of Chemical Ecotoxicology, UFZ Centre for Environmental Research Leipzig-Halle, Permoserstraße 15, 04318 Leipzig, Germany ‡Department of Analytical Chemistry, UFZ Centre for Environmental Research Leipzig-Halle, Permoserstraße 15, 04318 Leipzig, Germany ( Received 15 December 1999; Accepted 15 March 2000) Abstract—Within the scope of bioassay-directed identification of dioxin-like toxicants in complex environmental samples, EC50- based and fixed-effect-level–based 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity equivalents (TEQs) were compared to assess 7-ethoxyresorufin-O-deethylase (EROD) inducing potency of sediment fractions using the rainbow trout liver cell line RTL-W1 as bioassay system. Toxicity equivalents on the basis of fixed effect levels are suggested in order to minimize interpretation problems due to the superposition of enzyme-inducing and enzyme-inhibiting effects. Bioassay-directed fractionation of a contaminated sediment extract in the industrial region of Bitterfeld (Germany) based on fixed-effect-level TEQs indicated high dioxin-like activity in the lipophilic sediment fractions containing the prototypic arylhydrocarbon receptor (AhR) agonists polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). However, only a small part of the EROD induction could be attributed to the PCBs and PAHs that were analyzed. Significant EROD induction occurred also with some of the more polar fractions. Keywords—Ethoxyresorufin-O-deethylase Toxicity euivalent assessment Sediment extract Bioassay-directed fractionation Confirmation INTRODUCTION Despite bans and reduced emissions of some persistent or- ganic pollutants (POPs) (e.g., PCBs within the last decades), they are of undiminished high environmental relevance, and their environmental chemistry and ecotoxicology is a field of ongoing and extensive research. The high relevance of POPs is caused by their potential for long-range airborne transport to remote regions [1] and bioaccumulation that is driven by their generally high hydrophobicity and relatively high recal- citrance toward degradation under environmental conditions. Numerous POPs, such as polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), polychlorinated biphenyls (PCBs), po- lychlorinated naphthalenes (PCNs), but also polyaromatic hy- drocarbons (PAHs), are highly toxic, including carcinogenic and reproductive effects, thymic atrophy, body weight loss, and acute lethality in mammals. Many of these effects are thought to be mediated through the arylhydrocarbon receptor [2,3]. By binding to the AhR, dioxins, PCBs, and PAHs trigger a cascade of cellular events leading to increased expression of the cytochrome P4501A (CYP1A) gene and the associated catalytic 7-ethoxyresorufin-O-deethylase activity. The ability of certain classes of POPs to induce CYP1A is used as a basis for several bioanalytical procedures. Tillit et al. [4] used an in vitro EROD assay with H4IIE rat hepatoma cells to detect the presence of planar halogenated hydrocarbons in extracts from chicken eggs. Since the AhR pathway is phy- logenetically conserved [5], the EROD assay is not restricted to the use of mammalian cells but can be extended to cell systems of other taxa as well [6–12]. The use of nonmam- malian systems may offer insight into species-specific respons- es to POP exposure. Bioassay-directed chemical fractionation procedures, which * To whom correspondence may be addressed (wb@uoe.ufz.de). combine physicochemical fractionation steps with biotesting and chemical analysis [13,14], are an appropriate application of microbiotests, particularly when they indicate specific modes of toxic action and/or the presence of specific chemical classes. In vitro EROD assays in combination with physico- chemical fractionation and chemical analysis have been ap- plied to identify EROD-inducing compounds in environmental samples such as pulp mill effluents [15,16] and extracts of settling particulate matter [17]. A problem in the application of in vitro EROD assays for testing environmental samples, however, is the lack of a generally accepted approach to quan- tify EROD induction of complex environmental samples. This is related to the fact that complex samples containing mixtures of chemical compounds or fractions with complex matrices often do not result in classical, sigmoidal dose–response curves. Instead, EROD activity follows a complex bell-shaped dose–response relationship, with compound-dependent maxi- mum induction [6,18]. This may be the result of the super- position of enzyme induction and enzyme inhibition by the toxicants in the sample and makes it difficult to calculate re- liable EC50 values [7,10]. Several different endpoints have been used to estimate EROD induction potency of a sample, including absolute induction at a distinct concentration [15,16], EC50 values [4], or effect concentrations at a fixed effect level [19]. It remains to be clarified which of these endpoints is suitable for hazard assessment and toxicity iden- tification in environmental samples. One of the key questions in every toxicity identification study is whether the analyzed toxicants in the fractions can be confirmed to be responsible for the measured effects [20]. Because effects of complex environmental samples usually are not caused by one individual compound but by a mixture of toxicants, the toxicity confirmation requires a valid concept for joint toxicity. For EROD induction, the concept of con-